Radiotherapy combined with hypoxic cell sensitizers

ABSTRACT

The invention discloses a method of treating cancer in a patient, comprising administering to the patient a radiation sensitizer selected from nitroimidazoles in an amount effective to sensitize a patient to radiation and subjecting the patient to radiation therapy. In certain embodiments the radiation sensitizer is etanidazole or doranidazole.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. 371 ofInternational Application No. PCT/US2011/026674, filed Mar. 1, 2011,which claims the benefit of the filing date under 35 U.S.C. 119(e) toU.S. Provisional Application No. 61/309,388, filed Mar. 1, 2010. Thespecifications of each of the foregoing applications are herebyincorporated by reference in their entirety. International ApplicationPCT/US2011/026674 was published under PCT Article 21(2) in English.

BACKGROUND OF THE INVENTION

Cancer is one of the deadliest illnesses in the United States. Itaccounts for nearly 600,000 deaths annually, and costs billions ofdollars for those who suffer from the disease. This disease is in fact adiverse group of disorders, which can originate in almost any tissue ofthe body. In addition, cancers may be generated by multiple mechanismsincluding pathogenic infections, mutations, and environmental insults(see, e.g., Pratt et al., Hum. Pathol. 36:861-70, 2005). Current cancertreatments include, among others, surgery, chemotherapeutics, radiationtherapy, immunotherapy, and photodynamic therapy. However, none of thesetreatments is completely effective, and each has its own associated sideeffects.

Hypoxia is a characteristic feature of many tumors, particularly locallyadvanced and recurrent solid cancers resulting from an imbalance betweenoxygen supply and consumption (see Vaupel et al., Oncologist 9 Suppl.5:4-9, 2004). Cancer tumor hypoxia can reduce the effectiveness ofradiotherapy, some oxygen-dependent cytotoxic agents, and photodynamictherapy. The presence of hypoxia has been demonstrated in a wide varietyof human cancers, including colorectal, cervix, breast, lung, brain,pancreas, head and neck, and prostate. Many of these tumors containedregions of severe hypoxia (<5 mmHg oxygen). Clinically, the duration ofdisease and progression free survival correlates inversely with thedegree of tumor hypoxia. For example, in patients with squamouscarcinoma of the head and neck, the one-year disease-free survival was78% for patients with median tumor pO₂>10 mm Hg but only 22% for medianpO₂<10 mm (Brizel, et al., Int. J. Radiat. Oncol. Biol. Phys. 38:285-9,1997). Hypoxic cells exhibit increased resistance to standard radiationand chemotherapy treatment programs, as these cells are relativelyisolated from the blood supply and because radiation and chemotherapypreferentially kill rapidly dividing cell populations.

Pharmaceutical compounds which sensitize hypoxic cells to radiationtherapy have shown promising results. However, there remain drawbacks tothe safety and efficacy in the current methods.

SUMMARY OF THE INVENTION

The invention comprises a method of treating cancer in a patient,comprising administering to the patient a radiation sensitizer selectedfrom nitroimidazoles in an amount effective to sensitize a patient toradiation; and subjecting the patient to radiation therapy. In certainembodiments the radiation sensitizer is etanidazole or doranidazole.

In certain embodiments, the radiation therapy is intraoperativeradiation therapy (“IORT”). In particular embodiments, the radiation islocalized to a tumor site. The patient may be subjected tointraoperative radiation prior to resection of the tumor or followingresection of the tumor. The tumor site may comprise different types ofcells including cancerous and benign cells. In certain embodiments, theradiation therapy is stereotactic body radiotherapy (“SBRT”) orstereotactic radiosurgery (“SRS”).

The radiation may be ionizing radiation such as particle beam radiation.The particle beam radiation may be selected from any of electrons,protons, neutrons, heavy ions such as carbon ions, or pions. Theionizing radiation may be selected from x-rays, UV-light, γ-rays, ormicrowaves. In certain embodiments, the radiation therapy may comprisesubjecting the patient to one or more types of radiation therapy.

In certain embodiments, the method of the invention comprisesadministering radiation with a mobile electron beam therapy system. Theradiation may be delivered before, during or after a surgical procedure.In certain embodiments, the patient is administered a radiationsensitizer and subjected to radiation therapy within a short timethereafter, such as within about 2 hours of each other, such as withinabout 1 hour of each other, e.g., within about 40 minutes of each other.

In certain embodiments, the method of the invention is for the treatmentof cancer selected from colorectal cancer, stomach cancer, brain cancer,lung cancer, pancreatic cancer, prostate cancer, cancer of the head orneck, breast cancer, or cancer of the oral cavity. In particularembodiments the cancer is lung or colorectal. In certain embodiments,the method for treating cancer comprises: (a) administering to thepatient a pharmaceutically acceptable preparation which includes atherapeutically effective amount of a radiation sensitizer selected frometanidazole and doranidazole; and (b) subjecting the patient to atherapeutically effective amount of radiation.

In certain embodiments, the method for treating cancer comprises: (a)administering to the patient a radiation sensitizer selected frometanidazole and doranidazole in an amount effective to sensitize thepatient, e.g., the tumor, tumor bed and surrounding tissue, to ionizingradiation; and (b) subjecting the patient to intraoperative radiation.

In certain embodiments, the method comprises (a) administering to thepatient a pharmaceutically acceptable composition comprising atherapeutically effective amount of a radiation sensitizer; (b)performing resection of a tumor; and (c) subjecting the patient's bodycavity at the site of the resection of step (b) with a therapeuticallyeffective amount of intraoperative radiation therapy.

In certain aspects, the radiation sensitizer of the invention isassociated with a targeting moiety. The targeting moiety may be selectedfrom an antibody such as an antibody which targets a tumor-specificantigen. The targeting moiety may be covalently associated with theradiation sensitizer or the targeting moiety may be non-covalentlyassociated with a radiation sensitizer. The radiation sensitizer andtargeting moiety may be associated within a liposome.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION

Overview

Work has been going on for many years on methods for increasing theradiosensitivity of tumors relative to that of normal tissues. One ofthese methods involves administering a pharmaceutical that sensitizesthe tumor cells to radiation. The use of such pharmaceuticals, calledradiosensitizers, provides a method of increasing the radiosensitivityof tumors to radiation therapy, avoiding the need to increase radiationdosages to levels that are harmful to surrounding organs and tissues.

The largest class of radiosensitizers is the hypoxic cell sensitizers.These pharmaceuticals overcome the radioresistance afforded some tumorcells by their lack of oxygen, i.e., hypoxia. Most tumors exhibit somedegree of hypoxia, with locally advanced and recurrent tumors exhibitingespecially high levels of hypoxia. The decreased oxygenation of tumorcells is a consequence of the structural and functional disturbances tothe tumor vasculature that inhibit the normal delivery of oxygen, Withinthis class, electron-affinic nitroimidazoles have been found in generalto radiosensitize hypoxic tumor cells. Two nitroimidazoles, misonidazoleand metronidazole, have been used clinically; however, clinicalapplications are limited by neurotoxicity and lower dosages areineffective at sensitizing tumor cells to traditional external beamradiation therapy.

Etanidazole, an electron-affinic 2-nitroimidazole, displays asensitizing ability similar to misonidazole but has less lipophilicityand thus less neurotoxicity, allowing higher doses. Studies of themaximum tolerated dose of etanidazole have shown that 12 gm/m² maysafely be given to patients who receive single dose administrations.Above this level, the incidence of side effects increases in patientswho have received single dose administrations of etanidazole. Thus,methods of exploiting the reduced neurotoxicity of etanidazole forkilling hypoxic cells would be advantageous.

In traditional external beam radiation therapy coupled withradiosensitizer administration, a beam of high energy X-rays, generatedoutside the patient by a linear accelerator, is delivered to a tumor.Most body tissue does not absorb or block X-rays, so they progressthrough the body, constantly releasing energy. When the cancer tumor iswithin the path of the X-ray, it receives some of that radiation;however, surrounding healthy tissue receives radiation as well. In orderto limit the extent of collateral tissue damage, oncologists typicallybombard the tumor area with the lowest level of effective radiation frommany different points of entrance in an attempt to minimize damage tonormal tissues. Even modern external beam radiation systems withimproved real-time imaging of the patient anatomy will inevitably treatsubstantial normal tissue volumes when targeting the tumor.

Other energy sources, such as particle beams contain charged atomicparticles. Particle beams have tremendous energy but also high mass andas such they slow down as they encounter body tissue. Particles can becontrolled, for example, to release their energy at a specific point inthe body. Particle beam therapy uses electrons, neutrons, heavy ions(such as protons, carbon ions and helium); and pi-mesons (also calledpions).

Recent approaches to radiotherapy use high-dose radiation with precisefocus on the cancerous area, limiting exposure of healthy cells toradiation. Stereotactic Body Radiation Therapy (“SBRT”), usesimage-guided, focused high-dose external beam x-ray radiation toirradiate a tumor, often in a single fraction. To avoid the excessivetoxicity which can occur to normal tissue, however, many tumors, evenwhen targeted with SBRT, must be irradiated over two to five fractions,each fraction of lower dose than single fraction SBRT. The reduced doseper SBRT fraction may not be adequate to destroy the hypoxic componentof the tumor.

Stereotactic radiosurgery (“SRS”), is a non-surgical procedure thatdelivers a single high-dose of precisely-targeted radiation typicallytargeted to the brain, head and neck using highly focused gamma-ray orx-ray beams that converge on the specific area or areas where the tumorresides, minimizing the amount of radiation to healthy tissue. Althoughstereotactic radiosurgery is often completed in a one-day session,physicians sometimes recommend multiple treatments, especially fortumors larger than one inch in diameter. The procedure is usuallyreferred to as fractionated stereotactic radiosurgery when two to fivetreatments are given and as stereotactic radiotherapy when more thanfive treatments are given.

Intraoperative Radiation Therapy (“IORT”) is the delivery of radiationat the time of surgery using a focused high-dose radiation directed tothe site of the cancerous cells. IORT is characterized by a concentratedbeam of ionizing radiation to cancerous tumors while the patient isexposed during surgery, i.e., radiation is delivered within an open bodycavity. IORT has an advantage of being able to temporarily displacehealthy tissue from the path of the radiation beam so as to reduce theexposure of normal tissues to the radiation and contact the tumor sitemore directly. Single dose IORT in excess of 8-10 Gy, is effective atdestroying tumor stem cells and its host-derived microvascularstructure, thereby inhibiting DNA repair in the tumor, but hypoxic cellswithin the tumor may require doses in excess of 20-24 Gy, doses thatcould exceed normal tissue tolerance.

The present invention relates to methods of treating cancer in a patientcomprising administering to the patient a radiation sensitizer such as ahypoxic cell sensitizer and subjecting the patient to radiation therapy.In certain embodiments of the invention, coupling a radiation sensitizerwith radiation techniques that employ focused high-dose radiationtherapy increases the exposure of tumor cells to radiation whileprotecting the surrounding tissues and organs. The radiation sensitizermay be administered prior to the administration of this focusedhigh-dose radiation therapy, and due to the high local intensity of theradiation, the surrounding tissues and organs are spared the damage ofnon-directed radiation therapy. In one embodiment of the invention, apatient is administered a radiation sensitizer and then subjected toIORT. In another embodiment, administration of a radiation sensitizer isfollowed by SBRT or SRS. Such treatment may be used to treat any solidcancerous tumor, particularly tumors that are resistant to traditionaltherapies, such as locally advanced and recurrent head and neck tumorsand recurrent rectal cancer.

In certain embodiments, the radiation sensitizer is a nitroimidazole,such as a 2-nitroimidazole, e.g., etanidazole or doranidazole. Incertain embodiments, the radiation sensitizer is administered to apatient prior to or during a surgical procedure to remove a tumor. Insuch embodiments, the patient undergoes a surgical procedure to remove atumor and during the surgery, the patient is subjected to IORT such asduring or after the resection of the tumor. In certain embodiments, thepatient is subjected to intraoperative radiation more than once duringthe surgical procedure such as before the resection of the tumor andfollowing the tumor removal. In another embodiment, administration of anitroimidazole is followed by SBRT or SRS. The patient may beadministered the radiation sensitizer within 2 hours prior to beingsubjected to radiation therapy, such as within 1 hour or within 40minutes.

Definitions

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more.

As used herein, the phrase “conjoint administration” refers to any formof administration of two or more different therapeutic compounds suchthat the second compound is administered while the previouslyadministered therapeutic compound is still effective in the body (e.g.,the two compounds are simultaneously effective in the patient, which mayinclude synergistic effects of the two compounds). For example, thedifferent therapeutic compounds can be administered either in the sameformulation or in a separate formulation, either concomitantly orsequentially. Thus, an individual who receives such treatment canbenefit from a combined effect of different therapeutic compounds.

As used herein “fraction” or “fractionation” of radiation therapy isdividing the total dose of radiation therapy into several smaller dosesdelivered over a period of time. The total dosage may be fractionated toallow normal cells time to recover, to allow tumor cells that were in arelatively radio-resistant phase of the cell cycle during one treatmentto cycle into a sensitive phase of the cycle before the next fraction isgiven, or to allow hypoxic tumor cells to reoxygenate between fractions,improving the tumor cell kill. The summed value of individualfractionized dose should add up to about the total dose of radiationtherapy prescribed.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes prophylacticadministration of a composition which reduces the frequency of,decreases the severity of, or delays the onset of symptoms of a medicalcondition in a subject relative to a subject which did not receive thecomposition. Thus, prevention of cancer includes, for example, reducingthe number of detectable cancerous growths in a population of patientsreceiving a prophylactic treatment relative to an untreated controlpopulation, and/or delaying the appearance of detectable cancerousgrowths in a treated population versus an untreated control population,e.g., by a statistically and/or clinically significant amount.Prevention of an infection includes, for example, reducing the number ofdiagnoses of the infection in a treated population versus an untreatedcontrol population, and/or delaying the onset of symptoms of theinfection in a treated population versus an untreated controlpopulation. Prevention of pain includes, for example, reducing themagnitude of, or alternatively delaying, pain sensations experienced bysubjects in a treated population versus an untreated control population.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, (i.e., it protects thehost against developing the unwanted condition), whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

In the present invention, the term “radiation sensitizer” or“radiosensitizer” means a compound which enhances the effect ofradiation.

A “therapeutically effective amount” of a compound with respect to thesubject method of treatment refers to an amount of the compound(s) in apreparation which, when administered as part of a desired dosage regimen(to a mammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

As used herein, the term “treating” or “treatment” includes reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a subject'scondition.

As used herein, the term “surgery” is a medical technology consisting ofa physical intervention on tissues. A procedure is considered surgicalwhen it involves cutting of a patient's tissues or closure of apreviously sustained wound. Other procedures that do not necessarilyfall under this rubric, such as angioplasty or endoscopy, may beconsidered surgery if they involve common surgical procedure orsettings, such as use of a sterile environment, anesthesia, antisepticconditions, typical surgical instruments, and suturing or stapling.Surgery can last from minutes to hours, but is typically not an ongoingor periodic type of treatment. As used herein, “before surgery” refersto the period of time prior to the physical intervention on tissues,wherein intervention of tissues refers to cutting of a patient'stissues. Treatments administered before surgery may be administered inexemplary embodiments about 2 hours before, about 1 hour before, about45 minutes before, about 30 minutes before, about 20 minutes before orabout 10 minutes before the physical intervention on tissues. As usedherein, “during surgery” refers to the period of time after which thesurgical procedure has commenced, i.e., the physical intervention oftissues and continues until the time that the task being performedwithin the tissue is complete. For example, in the case where a cancerpatient is subjected to resection of a cancerous tumor, tissue isincised revealing the tumor, the tumor is removed during the surgicalprocedure, radiation may be administered to the patient and then thetissue is sutured, marking the completion of the surgery. Such radiationwould be considered to have been delivered during the surgery. Thecompletion of the surgical procedure is often marked by the closure oftissue through suturing or stapling.

Exemplary Embodiments

In certain embodiments, the invention provides a method of treatingcancer in a patient, comprising administering to the patient a radiationsensitizer selected from nitroimidazoles in an amount effective tosensitize a patient to radiation and subjecting the patient to radiationtherapy. Nitroimidazoles of the invention may be selected from anycompound with the characteristic features of a nitroimidazolefunctionality and which function as radiation sensitizers, such ashypoxic cell sensitizers. Exemplary nitroimidazoles of the inventioninclude etanidazole, doranidazole, metronidazole, misonidazole,tinidazole, nimorazole and compounds disclosed in U.S. Pat. No.4,282,232. In particular embodiments, the nitroimidazole is selectedfrom a 2-nitroimidazole such as etanidazole or doranidazole.

The methods of the invention may be used to treat any cancer, includingbut not limited, to a solid tumor, such as brain, lung, liver, spleen,kidney, lymph node, small intestine, pancreas, blood cells, bone, colon,rectum, stomach, breast, endometrium, prostate, testicle, ovary, centralnervous system, head, neck, or esophageal cancer. In certainembodiments, the methods of the invention are used to treat rectalcancer, lung cancer or cancer of the head and neck. In particularembodiments, the methods are used to treat rectal cancer. In particularembodiments, the methods are used to treat lung cancer.

In certain aspects the radiation therapy used to treat the patient isintraoperative radiation therapy (IORT). For example, the patient mayreceive a nitroimidazole radiation sensitizer and IORT while the patientis exposed during surgery, i.e., radiation is delivered within an openbody cavity. IORT may be delivered in a single dosage or fractionated intwo or multiple doses, e.g., in the duration of a surgical procedure.For example, the patient may be administered etanidazole or doranidazoleand subjected to IORT, e.g., within about 1 hour of each other, such aswithin about 40 minutes of each other. In particular embodiments, apatient suffering from rectal cancer is administered etanidazole andsubjected to IORT to treat the rectal cancer, e.g., within about 1 hourof each other.

For patients subjected to IORT, IORT may be administered at one or morestages during a surgical procedure. For example, the patient may beadministered a radiation sensitizer and surgically incised to reveal atumor, at which point radiation is administered directly to the tumor ora portion thereof. The radiation source may be placed in close proximityor in contact with the cancerous tissues or organs. Radiation may bedelivered to the patient with a mobile electron beam therapy system.

In certain embodiments, radiation therapy is localized to a tumor site.A patient may be subjected to radiation, e.g., IORT, prior to resectionof cancerous cells, e.g., a malignant tumor, such as about 1 hour prior,such as about 40 minutes prior to resection. Alternatively, the patientmay be subjected to radiation therapy following resection of cancerouscells, such as within about 1 hour of radiation therapy, or may even besubjected to radiation both prior to and following resection ofcancerous cells. In certain exemplary embodiments, the patient undergoessurgical resection of a tumor and radiation therapy is administered tothe patient during the surgical procedure, following removal of thetumor, or both during the surgical procedure and following the removalof the tumor. Thus, the method may comprise treating a tumor site withradiation, e.g., during a surgical procedure, after partial resection ofthe tumor. It would be well in the realm of knowledge of one of skill inthe art as to which of the tissues remaining after resection should betreated with radiation therapy. In certain embodiments, the patient iscontacted with one or more of IORT, SBRT or SRS at a tumor site.

Radiation localized to a tumor site may contact cancerous ornon-cancerous cells. In certain embodiments, the radiation localized tothe tumor site may contact non-cancerous cells, i.e., benign cells. Forexample, the method may comprise treating non-cancerous cellssurrounding a tumor site with radiation in order to prevent recurrenceof the cancer, e.g., through the irradiation of any microscopic diseasethat might extend into the normal tissue structures.

In certain embodiments, the surgical procedure is performed in closeproximity to the radiation source such that the patient does not need tobe moved during, before, or after the surgery to receive IORT. Forexample, the radiation source may be located in the operating room,e.g., to facilitate access to the radiation source during surgery. Incertain embodiments, the radiation is administered at one or more timesduring a surgical procedure.

A “surgical procedure” or “surgery” referred to in IORT includespreventative, diagnostic or staging, curative and palliative surgery.Curative surgery is a cancer treatment that may be used in conjunctionwith other therapies, such as the treatment of the present invention,chemotherapy, radiotherapy, hormonal therapy, gene therapy,immunotherapy and/or alternative therapies. Curative surgery includesresection in which all or part of cancerous tissue is physicallyremoved, excised, and/or destroyed. Tumor resection refers to physicalremoval of at least part of a tumor. In addition to tumor resection,treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and microscopically controlled surgery (Mohs' surgery).It is further contemplated that the present invention may also be usedin conjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

In certain embodiments, the radiation delivered with IORT is ionizing.Ionizing radiation may be particle beam radiation, also known as chargedparticle radiation, which uses beams of charged particles such aselectrons, protons (e.g., proton beam radiation), neutrons, pions, orcarbon ions. Ionizing radiation may also be selected from x-rays,UV-light, γ-rays or microwaves.

A combination of stereotactic radiation and a radiosensitizer may beused to sensitize the tumor cells and provide highly-focused doses ofradiation on the cells. In certain aspects, stereotactic radiation suchas SBRT or SRS is used in combination with a nitroimidazole radiationsensitizer, such as a 2-nitroimidazole, to treat cancer. In particularembodiments, the patient is administered a nitroimidazole radiationsensitizer, such as a 2-nitroimidazole, and subjected to SBRT, e.g.,within about 1 hour such as within about 40 minutes of each other. Incertain embodiments, the patient is administered a nitroimidazoleradiation sensitizer, such as a 2-nitroimidazole, and subjected to SRS,e.g., within about 1 hour such as within about 40 minutes of each other.In some embodiments, SBRT or SRS is delivered in a single dose or isfractionated in two or multiple doses such as over a period of hours,days or weeks. In other embodiments, SBRT or SRS is delivered from 2 ormore angles of exposure to intersect at the tumor, providing a largerabsorbed dose there than in the surrounding, healthy tissue. In stillother embodiments, SBRT or SRS is used with etanidazole or doranidazoleto treat cancer. In more particular embodiments, a patient sufferingfrom lung cancer is administered etanidazole and subjected to SBRT suchas within about 1 hour of each other.

The timing may be varied between the administration of a radiationsensitizer and radiation therapy. In certain aspects, the patient isadministered a radiation sensitizer and subjected to radiation therapy,e.g., IORT, SRS or SBRT, within about 2 hours of each other, such aswithin 1 hour, such as within 40 minutes, preferably such that the organor tissue to be irradiated has had adequate time to absorb a sufficientconcentration of the sensitizer prior to radiation treatment. Thus, thesensitizer may be administered less than 2 hours before radiationtreatment, or less than 1 hour such as about 80 minutes, about 70minutes, about 60 minutes, about 50 minutes, about 40 minutes, about 30minutes, about 20 minutes or about 10 minutes before radiationtreatment.

A radiation sensitizer, such as a 2-nitroimidazole, may be administeredto a patient before each fractionated dose of radiation is delivered,for example, within about 1 hour of each fractionated dose of radiation.In certain embodiments, the patient is administered etanidazole withabout 1 hour, for example, within about 40 minutes of each fractionizeddose of radiation therapy, such as SRS or SBRT.

A radiation sensitizer, such as 2-nitroimidazole, may be administered toa patient before each single dose of radiation is delivered, forexample, within about 1 hour of each single dose of radiation. Forexample, a patient may receive a single dose of IORT radiation during asurgical resection and a single dose of SBRT a day or more after thesurgery, both radiation doses of which may be preceded by administrationof a radiation sensitizer such as a 2-nitroimidazole. Each single dosemay be targeted to the same tumor site or different tumor sites. Incertain embodiments, two or more single radiation doses are targeted tothe same tumor site.

SBRT or SRS may be administered independently of other surgicalprocedures. In certain embodiments, the patient receives a single doseor fractionated doses of SBRT or SRS without undergoing any surgery.Patients may also receive one or more doses of SBRT or SRS, which may bepreceded by administration of a radiation sensitizer, such as2-nitroimidazole within, e.g., about 1 hour, of the one or more doses ofSBRT or SRS.

Radiation may be selected from any type suitable for treating cancer.Radiation may come from a machine outside the body (external radiation),may be placed inside the body (internal radiation), or may use unsealedradioactive materials that go throughout the body (systemic radiationtherapy). The type of radiation to be given depends on the type ofcancer, its location, how far into the body the radiation will need topenetrate, the patient's general health and medical history, whether thepatient will have other types of cancer treatment, and other factors. Incertain embodiments, radiation is delivered in more than one manner,e.g., internal radiation and external radiation.

One or more forms of radiation may be coupled with the radiationsensitizer of the invention. In certain embodiments, the patient isadministered a radiation sensitizer and subjected to a form of externalradiation and one or more additional forms of radiation. Externalradiation may be intraoperative electron beam radiation therapy, whichmay, for example, be administered during a surgical procedure, asdiscussed above. In particular embodiments, the patient is subjected toIORT and a second type of radiation selected from external, internal andsystemic radiation. In certain particular embodiments, the patient issubjected to intraoperative radiation therapy and external beamradiation therapy.

In those embodiments where the patient is subjected to more than oneform of radiation therapy, the patient may be subjected to two or moreforms of radiation therapy at the same time, in sequence, in fractionaldoses at the same time or in fractional doses sequentially, infractional doses alternating, and/or any combination thereof. In certainembodiments, intraoperative radiation therapy is administered before,during and/or after a surgical procedure and a second form or radiationtherapy is administered at a later time such as hours after the surgicalprocedure, and/or days after the surgical procedure, and/or weeks afterthe surgical procedure. In certain embodiments, the patient is treatedwith radiation therapy leading up to the surgical procedure such ashours before the surgical procedure, days before the surgical procedureand/or weeks before the surgical procedure.

Radiotherapy of the invention may comprise a cumulative externalirradiation of a patient in a dose of 1 to 100 Gy. A preferred range ofthe irradiation dose is 1 to 60 Gy. In certain embodiments, the dose ofradiation therapy is less than 90 Gy, such as less than 80 Gy, such asless than 70 Gy, such as less than 60 Gy, such as less than 50 Gy, suchas less than 40 Gy, such as less than 30 Gy, such as less than 20 Gy. Incertain embodiments the dose or radiation therapy is between about 10 to100 Gy, such as from about 20 to 80 Gy, such as about 30 to 70 Gy, suchas about 40 to 60 Gy. In certain embodiments, the irradiation dose isselected from 5-25 Gy, such as from 10-20 Gy.

An external irradiation dose may be administered in 1 to 60 fractionaldoses, such as from 5 to 30 fractional doses. In certain embodiments,the fractionized doses are administered with about 1.5 to about 2 Gy perfraction, such as about 1.5 Gy, such as about 1.6 Gy, such as about 1.7Gy, such as about 1.8 Gy, such as about 1.9 Gy, such as about 2.0 Gy,such as about 2.1 Gy, such as about 2.2 Gy, such as about 2.3 Gy such asabout 2.4 Gy, such as about 2.5 Gy per fractionized dose.

Fractionated doses of radiation therapy may be administered atintervals. In certain embodiments, the fractionized doses areadministered over a period of minutes, hours, or weeks such as 1 to 26weeks, such as from about 1 to 15 weeks, such as from 2 to 12 weeks. Incertain embodiments, the fractionized doses are administered over aperiod less than about 15 weeks, such as less than about 14 weeks suchas less than about 13 weeks, such as less than about 12 weeks, such asless than about 11 weeks, such as about less than about 10 weeks, suchas less than about 9 weeks, such as less than about 8 weeks, such asless than about 7 weeks, such as less than about 6 weeks, such as lessthan about 5 weeks, such as less than about 4 weeks. In certainembodiments, the cumulative external irradiation is a therapeuticallyeffective amount of radiation for killing cells.

In other embodiments, the radiation therapy is administered in a singledosage rather than in fractionized doses. For example, the single dosemay be administered with about 1-30 Gy per dose, such as from 5-20 Gy orsuch as about 10-15 Gy. IORT may be administered with a dose of about5-20 Gy. In certain embodiments, a radiation sensitizer is administeredto a patient and the patient is subjected to a single dose of radiationtherapy within 10 minutes, within 20 minutes, within 30 minutes, within40 minutes, within 50 minutes or within an hour of the administration ofthe sensitizer.

In some embodiments, the invention provides methods of administeringreduced dosages of radiation by combining intraoperative radiation withradiation sensitizers of the invention. Brown et al. (Int. J. RadiationOncology Biol. Phys., 2010, 78 (1): 323-327) provide modeling data infavor of using the radiosensitizer etanidazole (ET) in combination withstereotactic ablative radiotherapy (SABR). By calculating the expectedlevel of tumor cell killing following SABR, Brown et al. indicate thatadministration of ET prior to SABR will reduce the dose and frequency ofirradiation required to treat tumors and metastases, particularly intumors with high levels of hypoxia. Thus, in particular embodiments, theradiation is reduced by up to 1%, up to 5%, up to 10%, up to 15%, up to20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%,up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to85%, up to 90%, up to 95%, and up to 99% as compared to intraoperativeradiation therapy delivered without sensitizers. Moreover, the dosage ofradiation in intraoperative radiation therapy may reduce by 25-50%relative to the amount of external beam radiation therapy that may berequired to treat the disease without the sensitizer. Accordingly, theinvention also provides a method of administering reduced dosages ofradiation to a patient by combining IORT, SBRT or SRS with a radiationsensitizer selected from nitroimidazoles, wherein the radiation isreduced by up to 75% relative to radiation therapy delivered withoutsensitizers.

The energy source used for the radiation therapy may be selected fromX-rays or gamma rays, which are both forms of electromagnetic radiation.X-rays are created by machines called linear accelerators. Depending onthe amount of energy the x-rays have, they can be used to destroy cancercells on the surface of the body, i.e., lower energy, or deeper intotissues and organs, i.e., higher energy. Compared with other types ofradiation, x-rays can deliver radiation to a relatively large area.Gamma rays are produced when isotopes of certain elements, such asiridium and cobalt 60, release radiation energy as they decay. Eachelement decays at a specific rate and each gives off a different amountof energy, which affects how deeply it can penetrate into the body.Gamma rays produced by the decay of cobalt 60 are used in the treatmentcalled the “gamma knife.”

The energy source for the radiation therapy may be selected fromparticle beams, which use fast-moving subatomic particles instead ofphotons. This type of radiation may be referred to as particle beamradiation therapy or particulate radiation. Particle beams may becreated by linear accelerators, synchrotrons, betatrons and cyclotrons,which produce and accelerate the particles required for this type ofradiation therapy. Particle beam therapy may use electrons, which areproduced by an x-ray tube, this may be called electron-beam radiation;neutrons, which are produced by radioactive elements and specialequipment; heavy ions such as protons, carbon ions and helium; andpi-mesons, also called pions, which are small, negatively chargedparticles produced by an accelerator and a system of magnets. Unlikex-rays and gamma rays, some particle beams, depending on the energy, canpenetrate only a short distance into tissue. Therefore, they are oftenused to treat cancers located on the surface of or just below the skin.

In the present invention, the term “ionizing radiation” means radiationcomprising particles or photons that have sufficient energy or canproduce sufficient energy via nuclear interactions to produceionization, i.e., gain or loss of electrons. The amount of ionizingradiation needed to kill a given cell generally depends on the nature ofthat cell. Means for determining an effective amount of radiation arewell known in the art. Used herein, the term “an effective dose” ofionizing radiation means a dose of ionizing radiation that produces anincrease in cell damage or death when given in conjunction with thenitroimidazoles of the invention.

In certain embodiments, the radiation therapy comprises ionizingradiation, particularly electron beam radiation. An electron beam may bedelivered intraoperatively to the tumor site using an electron beamtherapy system such as the one described in U.S. Pat. Nos. 5,418,372 and5,321,271 the full disclosure of which is incorporated herein byreference. In particular embodiments, the electron beam therapy systemof the invention provides adequate shielding to healthy tissue forprimary x-rays generated by the system as well as for scatter radiation.

In particular embodiments, the particle beam therapy is proton beamtherapy. Protons deposit their energy over a very small volume, which iscalled the Bragg peak. The Bragg peak can be used to target high dosesof proton beam therapy to a tumor while doing less damage to normaltissues in front of and behind the tumor. Proton beam therapy isgenerally reserved for cancers that are difficult or dangerous to treatwith surgery, such as a chondrosarcoma at the base of the skull, or itis combined with other types of radiation. Proton beam therapy is alsobeing used in clinical trials for intraocular melanoma, i.e., melanomathat begins in the eye, retinoblastoma, i.e., an eye cancer that mostoften occurs in children under age 5, rhabdomyosarcoma, i.e., a tumor ofthe muscle tissue, some cancers of the head and neck, and cancers of theprostate, brain, and lung.

In some embodiments, the radiation therapy is stereotactic (orstereotaxic) radiosurgery which uses a large dose of radiation todestroy tumor tissue. In certain exemplary embodiments, where the canceris in the brain, the patient's head can be placed in a special frame,which is attached or is fitted to the patient's skull. The frame is usedto aim high-dose radiation beams directly at the tumor inside thepatient's head. The dose and area receiving the radiation arecoordinated very precisely resulting in little damage to nearby tissues.In some stereotactic applications, a head frame is not needed. Incertain embodiments, real-time imaging systems are used in conjunctionwith the movement of the accelerator, allowing computer adjustments ofthe accelerator trajectory to compensate for any motion of the patient'shead.

Stereotactic radiosurgery may be done in a variety of ways. One suitabletechnique uses a linear accelerator to administer high-energy photonradiation to the tumor, i.e., linac-based stereotactic radiosurgery. Inanother technique, a gamma knife uses cobalt 60 to deliver radiation. Ina third technique, heavy charged particle beams such as protons andhelium ions are used to deliver stereotactic radiation to the tumor.

In certain embodiments, stereotactic radiosurgery is used in thetreatment of small benign and malignant tumors such as brain tumors,e.g., meningiomas, acoustic neuromas, and pituitary cancer. In addition,stereotactic radiosurgery can be used to treat metastatic brain tumors,i.e., cancer that has spread to the brain from another part of the bodyeither alone or along with whole-brain radiation therapy. Whole-brainradiation therapy is a form of external radiation therapy that treatsthe entire brain with radiation.

Radiation therapy may be stereotactic body radiotherapy, or SBRT.Stereotactic radiotherapy uses essentially the same approach asstereotactic radiosurgery to deliver radiation to the target tissue;however, stereotactic radiotherapy generally uses multiple smallfractions of radiation as opposed to one large dose, but certainapplications of SBRT may still be accomplished with a single fraction.Stereotactic body radiotherapy may be used to treat tumors in the brain,lung, liver, pancreas, prostate, spine, as well as other parts of thebody.

When a source of radiation therapy is internal, the energy used ininternal radiation may come from a variety of sources. For example, theradioactive isotope may be radioactive iodine, e.g., iodine 125 oriodine 131, strontium 89, phosphorous, palladium, cesium, iridium,phosphate, cobalt, or any other isotope known in the art. In certainembodiments, the internal radiation is administered as brachytherapy, aradiation treatment based on implanted radioactive seeds emittingradiation from each seed.

Radiation may be delivered directly to the cancer through the use ofradiolabeled antibodies, i.e., radioimmunotherapy. Antibodies are highlyspecific proteins that are made by the body in response to the presenceof antigens, i.e., substances recognized as foreign by the immunesystem. Some tumor cells contain specific antigens that trigger theproduction of tumor-specific antibodies. Large quantities of theseantibodies can be made in the laboratory and attached to radioactivesubstances, a process known as radiolabeling. Once injected into thebody, the antibodies seek out cancer cells, which are destroyed by theradiation. This approach can reduce or minimize the risk of radiationdamage to healthy cells. In certain embodiments, the radioimmunotherapytreatments are selected from ibritumomab tiuxetan (Zevalin®) andtositumomab and iodine 131 tositumomab (Bexxar®). Radioimmunotherapy maybe used in the treatment of advanced adult non-Hodgkin lymphoma (NHL).In certain embodiments, immunotherapy is used in the treatment ofcancers including leukemia, NHL, colorectal cancer, and cancers of theliver, lung, brain, prostate, thyroid, breast, ovary, and pancreas.

In certain aspects, the invention comprises the methods for planningexternal radiation therapy in order to target the cancerous cells andlimit exposure to healthy cells. In certain embodiments, the planning ofradiation treatments is performed in two dimensions (width and height)or three dimensions, for example, with three-dimensional (3-D) conformalradiation therapy. In certain embodiments, 3-D conformal radiationtherapy uses computer technology to allow doctors to more preciselytarget a tumor with radiation beams (using width, height, and depth). A3-D image of a tumor can be obtained using computed tomography (CT),magnetic resonance imaging (MRI), positron emission tomography (PET), orsingle photon emission computed tomography (SPECT). Using informationfrom the image, special computer programs may design radiation beamsthat “conform” to the shape of the tumor. In certain embodiments,because the healthy tissue surrounding the tumor is largely spared bythis technique, higher doses of radiation can be used to treat thecancer. Improved outcomes with less toxicity with 3-D conformalradiation therapy may be possible for nasopharyngeal, prostate, lung,liver, and brain cancers.

In certain particular embodiments, the radiation therapy isintensity-modulated radiation therapy (IMRT). IMRT is a type of 3-Dconformal radiation therapy that uses radiation beams, e.g., x-rays ofvarying intensities to deliver different doses of radiation to smallareas of tissue at the same time. The technology allows for the deliveryof higher doses of radiation within the tumor and lower doses to nearbyhealthy tissue. Some techniques deliver a higher dose of radiation tothe patient each day, potentially shortening the overall treatment timeand improving the success of the treatment. IMRT may also lead to fewerside effects during treatment. In particular embodiments, the radiationis delivered by a linear accelerator that is equipped with a multileafcollimator (a collimator helps to shape or sculpt the beams ofradiation). The equipment can be rotated around the patient so thatradiation beams can be sent from the best angles. The beams conform asclosely as possible to the shape of the tumor. In certain embodiments,this technology is used to treat tumors in the brain, head and neck,nasopharynx, breast, liver, lung, prostate, and uterus.

Radiation therapy may be used in conjunction with hyperthermia, i.e.,the use of heat. In certain embodiments, the combination of heat andradiation can increase the response rate of some tumors.

A radiosensitizer may be administered in conjunction with an additionalagent. For example, a nitroimidazole may be administered in conjunctionwith an additional agent such as a targeting agent, a chemotherapeuticagent or a second radiosensitizer. Targeting agents include any suitableagents for targeting cancer cells, such as antibodies. Nitroimidazolemay be bound to the targeting agent through covalent or non-covalentattachments. For example, nitroimidazoles, such as 2-nitroimidazoles,may be bound to a targeting agent through a linker such as abiodegradable linker. Alternatively, the nitroimidazoles may be bound toa targeting agent through ionic interactions. In certain embodiments,the radiosensitizer of the invention and the additional agent may beenveloped within a liposome.

In certain embodiments, the radiation sensitizer of the invention isassociated with a targeting moiety. The targeting moiety may becovalently bound to the nitroimidazole, or associated with thenitroimidazole though non-covalent forces such as ionic bonds, hydrogenbonds, or via encapsulation within a liposome. The targeting moiety,which assists the nitroimidazole in localizing to a particular targetregion, entering a target tumor cell(s), and/or locating within orproximal to the cell, may be selected on the basis of the particularcell type to be targeted. The targeting moiety may further comprise anyof a number of different chemical entities. In one embodiment, thetargeting moiety is a small molecule. Molecules which may be suitablefor use as targeting moieties in the present invention include haptens,epitopes, and dsDNA fragments and analogs and derivatives thereof. Suchmoieties bind specifically to antibodies, fragments or analogs thereof,including mimetics (for haptens and epitopes), and zinc finger proteins(for dsDNA fragments). Nutrients believed to trigger receptor-mediatedendocytosis and therefore useful targeting moieties include biotin,folate, riboflavin, carnitine, inositol, lipoic acid, niacin,pantothenic acid, thiamin, pyridoxal, ascorbic acid, and the lipidsoluble vitamins A, D, E and K. Another exemplary type of small moleculetargeting moiety includes steroidal lipids, such as cholesterol, andsteroidal hormones, such as estradiol, testosterone, etc.

Targeting moieties may also comprise one or more proteins. Particulartypes of proteins may be selected based on known characteristics of thetarget site or target cells. For example, the probe can be an antibodyeither monoclonal or polyclonal, where a corresponding antigen isdisplayed at the target site. In situations wherein a certain receptoris expressed by the target cells, the targeting moiety may comprise aprotein or peptidomimetic ligand capable of binding to that receptor.Proteins ligands of known cell surface receptors include low densitylipoproteins, transferrin, insulin, fibrinolytic enzymes, anti-HER2,platelet binding proteins such as annexins, and biological responsemodifiers (including interleukin, interferon, erythropoietin andcolony-stimulating factor). A number of monoclonal antibodies that bindto a specific type of cell have been developed, including monoclonalantibodies specific for tumor-associated antigens in humans. Among themany such monoclonal antibodies that may be used are anti-TAC, or otherinterleukin-2 receptor antibodies; 9.2.27 and NR-ML-05 to the 250kilodalton human melanoma-associated proteoglycan; and NR-LU-10 to apancarcinoma glycoprotein. An antibody employed in the present inventionmay be an intact (whole) molecule, a fragment thereof, or a functionalequivalent thereof. Examples of antibody fragments are F(ab′)₂, Fab′,Fab, and F_(v) fragments, which may be produced by conventional methodsor by genetic or protein engineering.

Other preferred targeting moieties include sugars, e.g., glucose,fucose, galactose, mannose, that are recognized by target-specificreceptors. For example, instant claimed constructs can be glycosylatedwith mannose residues, e.g., attached as C-glycosides to a freenitrogen, to yield targeted constructs having higher affinity binding totumors expressing mannose receptors, e.g., glioblastomas andgangliocytomas, and bacteria, which are also known to express mannosereceptors (Bertozzi, C R and M D Bednarski Carbohydrate Research 223:243(1992); J. Am. Chem. Soc. 114:2242, 5543 (1992)), as well as potentiallyother infectious moieties. Certain cells, such as malignant cells andblood cells (e.g., A, AB, B, etc.) display particular carbohydrates, forwhich a corresponding lectin may serve as a targeting moiety.

In certain embodiments, chemotherapeutic agents may be administeredconjointly with the methods of the invention. Chemotherapeutic agentinclude those compounds with anti-cancer activity, e.g., compounds thatinduce apoptosis, compounds that reduce lifespan or compounds thatrender cells sensitive to stress and include: aminoglutethimide,amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin,buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine,chlorambucil, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, dienestrol, diethylstilbestrol, docetaxel, doxorubicin,epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin,leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen,temozolomide, teniposide, testosterone, thioguanine, thiotepa,titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine,vincristine, vindesine, and vinorelbine.

These chemotherapeutic agents may be categorized by their mechanism ofaction into, for example, following groups: anti-metabolites/anti-canceragents, such as pyrimidine analogs (5-fluorouracil, floxuridine,capecitabine, gemcitabine and cytarabine) and purine analogs, folateantagonists and related inhibitors (mercaptopurine, thioguanine,pentostatin and 2-chlorodeoxyadenosine (cladribine));antiproliferative/antimitotic agents including natural products such asvinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubuledisruptors such as taxanes (paclitaxel, docetaxel), vincristine,vinblastine, nocodazole, epothilones, and navelbine,epidipodophyllotoxins (teniposide), DNA damaging agents (actinomycin,amsacrine, anthracyclines, bleomycin, busulfan, camptothecin,carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan,dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin,hexamethylmelamine, oxaliplatin, iphosphamide, melphalan,merchlorethamine, mitomycin, mitoxantrone, nitrosourea, paclitaxel,plicamycin, procarbazine, teniposide, triethylenethiophosphoramide andetoposide (VP16)); antibiotics such as dactinomycin (actinomycin D),daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines,mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase which systemically metabolizes L-asparagine anddeprives cells which do not have the capacity to synthesize their ownasparagine); antiplatelet agents; antiproliferative/antimitoticalkylating agents such as nitrogen mustards (mechlorethamine,cyclophosphamide and analogs, melphalan, chlorambucil), ethyleniminesand methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates(busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin),trazenes (e.g., dacarbazinine (DTIC)); antiproliferative/antimitoticantimetabolites such as folic acid analogs (methotrexate); platinumcoordination complexes (cisplatin, carboplatin), procarbazine,hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs(estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromataseinhibitors (letrozole, anastrozole); anticoagulants (heparin, syntheticheparin salts and other inhibitors of thrombin); fibrinolytic agents(such as tissue plasminogen activator, streptokinase and urokinase),aspirin, COX-2 inhibitors, dipyridamole, ticlopidine, clopidogrel,abciximab; antimigratory agents; antisecretory agents (breveldin);immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus(rapamycin), azathioprine, mycophenolate mofetil); anti-angiogeniccompounds (TNP-470, genistein) and growth factor inhibitors (vascularendothelial growth factor (VEGF) inhibitors, fibroblast growth factor(FGF) inhibitors, epidermal growth factor (EGF) inhibitors); angiotensinreceptor blocker; nitric oxide donors; anti-sense oligonucleotides;antibodies (trastuzumab); cell cycle inhibitors and differentiationinducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors(doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin,dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan(CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids(cortisone, dexamethasone, hydrocortisone, methylprednisolone,prednisone, and prednisolone); growth factor signal transduction kinaseinhibitors; mitochondrial dysfunction inducers and caspase activators;chromatin disruptors.

Radioprotectors may be administered to a patient in combination with themethods described herein. Radioprotectors, also called radioprotectants,are drugs that protect normal (noncancerous) cells from the damagecaused by radiation therapy. These agents promote the repair of normalcells that are exposed to radiation. Exemplary radioprotectants includeAmifostine (trade name Ethyol®).

In certain embodiments, the methods of the invention further compriseadministration of a bacterium such as salmonella or geneticallyengineered variants thereof. Studies have shown that the combination ofradiation therapy with salmonella increases the effectiveness of tumorsuppression particularly in the presence of inflammatory cells calledneutrophils. Therapies which combine a nitroimidazoles with a bacteriumsuch as salmonella and radiotherapy may enhance tumor suppression.

Radiation sensitizers of the invention may be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients. For example, compounds of the invention andtheir physiologically acceptable salts and solvates may be formulatedfor administration by, for example, injection (e.g. subcutaneous,intramuscular, intraparenteral), inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, sublingual, transdermal,nasal, parenteral or rectal administration. In one embodiment, acompound of the invention may be administered locally, at the site wherethe tumor cells are present, i.e., in a specific tissue, organ, or fluid(e.g., blood, cerebrospinal fluid, etc.). Typically, compounds of theinvention are administered intravenously.

The phrase “pharmaceutically acceptable” or “physiologically acceptable”is employed herein to refer to those ligands, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Radiation sensitizers of the invention can be formulated for a varietyof modes of administration. Techniques and formulations generally may befound in Remington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa.

In certain embodiments, pharmaceutical compositions may comprise atherapeutically effective amount of a nitroimidazole, for example, atleast about 0.1% of a nitroimidazole compound. In other embodiments, thean active compound may comprise between about 2% to about 75% of theweight of the unit, or between about 25% to about 60%, for example, andany range derivable therein. In other non-limiting examples, a dose mayalso comprise from about 0.1 mg/kg/body weight, 0.5 mg/kg/body weight, 1mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/bodyweight, about 20 mg/kg/body weight, about 30 mg/kg/body weight, about 40mg/kg/body weight, about 50 mg/kg/body weight, about 75 mg/kg/bodyweight, about 100 mg/kg/body weight, about 200 mg/kg/body weight, about350 mg/kg/body weight, about 500 mg/kg/body weight, about 750 mg/kg/bodyweight, to about 1000 mg/kg/body weight or more per administration, andany range derivable therein. In non-limiting examples of a derivablerange from the numbers listed herein, a range of about 10 mg/kg/bodyweight to about 100 mg/kg/body weight, etc., can be administered, basedon the numbers described above.

Toxicity and therapeutic efficacy of compounds of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals. The LD₅₀ is the dose lethal to 50% of thepopulation. The ED₅₀ is the dose therapeutically effective in 50% of thepopulation. The dose ratio between toxic and therapeutic effects(LD₅₀/ED₅₀) is the therapeutic index. Radiation sensitizers of theinvention that exhibit large therapeutic indexes are preferred. Whileradiation sensitizers of the invention that exhibit toxic side effectsmay be used, care should be taken to design a delivery system thattargets such compounds to the site of affected tissue in order tominimize potential damage to uninfected cells and, thereby, reduce sideeffects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds may lie within a range of circulating concentrations thatinclude the ED₅₀ with little or no toxicity. The dosage may vary withinthis range depending upon the dosage form employed and the route ofadministration utilized. For any compound, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

A pharmaceutical composition as described herein may comprise variousantioxidants to retard oxidation of one or more component. Additionally,the prevention of the action of microorganisms can be brought about bypreservatives such as various antibacterial and antifungal agents,including, but not limited to parabens (e.g., methylparabens,propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal orcombinations thereof.

The nitroimidazole, e.g., 2-nitroimidazole, may be formulated into acomposition in a free base, neutral or salt form. Pharmaceuticallyacceptable salts include the salts formed with a free carboxyl group oramine group derived from inorganic bases such as for example, sodium,potassium, ammonium, calcium or ferric hydroxides; or such organic basesas isopropylamine, trimethylamine, histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising, but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc.), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount of the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents that delay absorption, such as, for example, aluminummonostearate, gelatin or combinations thereof.

EXAMPLES

Overview

The following example describes an evaluation of the effectiveness ofIORT with and without the hypoxic cell radiosensitizer etanidazole forpatients with locally advanced primary or locally recurrent colorectalcarcinoma (also receiving chemotherapy and external beam irradiation).Results include: (a) percentage and length of local control of disease,(b) disease-free interval and overall survival time in all patients withevaluation of mode and cause of death, and (c) interval to failure andsite of failure (local, regional and/or distant).

Patient Selection

All patients meet the following criteria:

(1) Patients are in suitable medical condition to tolerate an operativeprocedure since an attempt at total or subtotal resection before orafter external irradiation is preferred in all patients.

(2) Patients have localized biopsy-proven recurrent (tumor bed orregional nodes) or primary locally advanced carcinoma of the rectum orcolon (cecum, ascending, transverse, descending or sigmoid), withoutevidence of distant metastases (peritoneal or blood borne).

(3) Patients have adequate bone marrow function and peripheralhematologic values with a hemoglobin of more than 10 mg/dl, white bloodcount of equal to or >40000/mm³ and platelets equal to or >100.000/mm³.They have adequate renal function as evidenced by a BUN of < or equal to30 mg/dl, or a creatinine of < or equal to 1.5 mg/dl, creatinineclearance of equal to or >50 ml/min., LFT's of <2× normal (bilirubin,SGOT, SGPT, alkaline phosphatase).

(4) If >⅓ of kidney would be within an irradiation field for extrapelvicdisease, bilateral renal function is demonstrated on an excretoryurogram (IVP), abdominal CT or renal scan.

(5) Patients have a Karnofsky performance status of >60%.

(6) Patients receiving prior treatment with 5-FU based adjuvant therapyare eligible for this protocol if the drug was not discontinued becauseof disease progression.

(7) Orthovoltage and HDR-IORT institutions thickness of residual diseasemust be <1 cm.

Patients are separated into 2 groups.

(1) Group A (no previous EBRT). Patients in Group A receive chemotherapyand standard preoperative irradiation to 45-50.4 Gy. At the time ofsubsequent exploration and/or resection, or on the day of surgery, thepatient is randomized. Treatment is given per the arm assigned. Patientsin Group A are randomized between Arm 2-IORT boost of 12.5-20 Gy totumor bed or unresected tumor alone, or Arm 3-IORT boost of 12.5-20 Gywith etanidazole. Patients undergo surgery/IORT by week 8 followingcompletion of initial chemotherapy and external irradiation. Therandomization takes place intraoperatively once it is known of patientsare study candidates, but randomization may be carried out earlier onthe day of surgery.

(2) Group B (Previous EBERT). Patients in Group B are registered onstudy and given at least low-dose preop irradiation (20-40 Gy) withcontinuous infusion 5-FU if safely feasible (Section 6.1.2). On the dayof surgery, or preferably the time of exploration/resection, if an IORTboost is feasible, the patient is immediately randomized (Step 2)between Arm 4 intraoperative radiation therapy boost of 15-20 Gy aloneor Arm 5-same IORT boost with etanidazole. At the time of surgery andIORT, a maximum resection is carried out, followed by reconstruction.

Radiation Therapy

Patients eligible for the protocol will be treated with preoperativeEBRT and chemotherapy combinations and techniques currently recommendedby the National Community Cancer Network. If postoperative radiation isrequired, that will also be applied in accordance with current NCCNrecommendations

Dose Time Factors and Interval from Operation to External BeamRadiation. In the majority of patients, one should be able to startexternal beam irradiation within 2-4 weeks of the most recent operation.Suggested intervals by type of operation are as follows: Exploratoryonly or resection with no bowel or gastric resection—≧2 weeks; Largebowel anastomosis +/−/small bowel-usually 3-4 weeks (occasionally up to6 weeks); abdominoperineal resection—usually 3-4 weeks (occasionally upto 8 weeks).

External Beam Large Field Component. For Group A, (no prior EBRT), thedose delivered to the extended tumor bedp—nodal portal is 45-50.4 Gy/5-6weeks (1.80 Gy/day, two or more fields per day, 5 days) per weekpreferably given preoperatively. For Group B (previous EBRT) patientsare retreated with a dose of 20-40 Gy preoperatively, 1.8-2 Gy/day (or1.2 Gy b.i.d) with PV15-FU.

Boost Field. When the extended field is limited to 45 Gy, an additional5.4 Gy should be delivered, whenever feasible, to a reduced field in 1.8Gy daily fractions (for a total of 50.4 Gy). Boost fields are treatedwith multi-field techniques or paired laterals. For pelvic lesions thismay include 3-field (PA and laterals), 4-field (lateral and pairedposterior obliques, PA-AP and laterals) or non-coplanar techniques. Withextrapelvic primaries, treatment in decubitus position with shapedlateral boost portals is often helpful in deleting additional smallintestine.

For extra pelvic disease, multiple field techniques may also be feasiblefor the boost dependent on the location of the disease. If residualinvolves 1) pancreas: 4-field boost, AP plus laterals, non-coplanarbeams; 2) posterior iliac fossa or posterior abdominal wall: PA pluslateral, etc.

Radiation Checklist. During irradiation, patients are seen in statuscheck at least once a week with notation of tolerance, weight and bloodcounts. Blood counts are obtained at least twice a week until past thenadir and then weekly. If the WBC subsequently falls below 3000 orplatelet count below 75,000, twice a week counts should be resumed. Ifthe WBC falls below 2000 or the platelet count below 50,000 during thecourse of irradiation, treatment should be delayed until the counts riseabove these levels.

Treatment port films are obtained for each treatment field and madeavailable for review. Frequency of port films of each field is at leastevery other week.

RT Treatment Interruptions. Interruptions which are required because ofRT-related toxicity or chemotherapy toxicity or major holidays do notresult in protocol deviation. Regardless of cause, interruptions whichprolong treatment by <10% do not result in protocol variation.Interruptions which prolong treatment by ≧10% constitute a minorprotocol variation.

Intraoperative Irradiation. IORT with Electrons (IOERT).

The maximum acceptable tumor thickness (depth) is 5.5 cm. In mostinstances the lesion is resected before or after the external beamcomponent of irradiation. The area of residual or unresected diseaseplus a minimum 1-1.5 cm. margin is included within single or abuttingintraoperative fields.

IOERT Dose. Doses are specified at the 100% isodose line. The entiretumor is encompassed by the 90% isodose line. Electron energies chosendepends on thickness of tumor bed or unresected tumor (after resectionthe common energy range is 6-12 MeV and without resection 15-18 MeV),and degree of beam obliquity.

Dose delivered to the 90% isodose depends on amount of remainingdisease:

Group A:

1) Resected, recurrent patients only, no residual or microscopicresidual-12.5-15 Gy (Given Dose [GD] 13.75-16.5 Gy)

2) Resected, gross residual equal to or less than 2 cm. maximumdimension −15 Gy (GD 16.5 Gy)

3) Unresected or gross residual greater than or equal to 2 cm.−17.5 to20 Gy (GD 19.25-22 Gy).

Group B:

1) Resected, no residual or microscopic residual-15 to 20 Gy (GD 16.5 to22 Gy).

2) Unresected or gross residual: 20 Gy (GD 22 Gy).

If a patient has an area of microscopic residual adjacent to grossresidual disease, it is desirable to use a shrinking field IOERTtechnique (i.e., deliver 13.75-16.5 Gy GD to a large field encompassingboth gross and microscopic residual; then use reduced cone size orshielding to deliver additional dose to the area of gross residual).

A number of radiation options exist to facilitate accurate delivery ofpelvic intraoperative irradiation. These are 1) use of applicators withbeveled ends, 2) use of perineal as well as abdominal approaches withprone as well as supine positioning of the patient, 3) increasedelectron energy if degree of beam obliquity suggests risk ofunderdosage, 4) use of beam shaping, or when possible, a lower electronenergy to decrease volume of a dose limiting organ within theintraoperative field. When possible, uninvolved dose limiting structureswhich cannot be physically excluded from the treatment field is shieldedsecondarily with malleable sterilized lead sheets of a thicknessappropriate to reduce the dose by 90%.

The maximum acceptable tumor thickness is 1 cm.

Doses are the same as for electron IORT with regard to dose per amountof residual (maximum dimension).

Drug Therapy

Drug therapy will be provided in accordance with current NCCN guidelinesfor preoperative and adjuvant chemotherapy for locally advanced andrecurrent rectal cancer.

Drug Information-Etanidazole (NSC#301467) (NCI IND#21,301). Dosage ofetanidazole: the dose of etanidazole to be administered is 12 gm/m².(This requires in the range of 500 cc of etanidazole in solution.)

Timing of IORT Radiotherapy.

Etanidazole is administered as a running intravenous infusion over a 15minute period. The surgeon and radiation oncologist determine when it is45 minutes prior to IORT. Etanidazole infusion begins at that time.Intraoperative radiotherapy is administered no earlier than 20 minutesafter the completion of the infusion (equal to or >35 minutes after thestart of the infusion). The timing of drug administration starting withtime zero as the start of the infusion is recorded. The interval betweenthe completion of drug infusion and IORT is recorded. The duration ofdrug infusion is also recorded.

The etanidazole solution is infused in a running intravenous solutionover a 15 minute period; the duration of drug infusion must be recorded.Blood pressure is carefully monitored during administration, ashypotension may be a side effect.

Surgery

A. Operative Procedures. An adequate procedure is one which allowsexploration of the entire abdomen as well as removal of known grosstumor with accompanying mesenteric lymph nodes whenever possible. Forrectal lesions a perineal approach alone is not acceptable. Acceptableprocedures, therefore, include variants of anterior resection,abdominal-perineal resection or Hartman procedure with end colostomy perthe discretion of the operating surgeon. When abdominal-perinealresection is necessary, some form of primary closure of the perineumshould be used unless problems with hemostasis exist.

For the purpose of external irradiation following surgery and/ordetermination of local failure patterns, all margins of tumor ortreatment volume are marked with small hemoclips (anterior, superior,inferior and both lateral margins; posterior if applicable). If externalirradiation has already been given (Group B), as much gross disease aspossible are resected and the full area of recurrent disease marked withhemoclips. Supine and crosstable lateral films are taken and reviewedwith the radiation oncologist for the purpose of treatment planningearly in the postoperative period. In addition, management of otherorgans with cancer involvement, i.e., ureter, bladder, uterus and ovary,are handled by standard surgical techniques. This is left to thediscretion of the operating surgeon although documentation of theprocedure performed must be provided.

B. Operative Procedure With Respect to IORT. The intent of operation isto 1) expose the tumor or tumor bed, 2) resect gross residual if at allpossible, and 3) minimize the amount of normal tissue and organs withinthe intraoperative irradiation field. In most institutions, it should bepossible to do the resection and deliver the IORT on the same day, withtemporary wound closure during transport of patients from the operatingroom to radiotherapy when indicated.

At some institutions, a second operation may be necessary for thepurpose of delivering IORT. In such instances, major resections may needto be performed in the hospital operating room suites with reoperationin the radiation oncology area within 1-7 days for the IORT.

After adequate exposure and a gross total or subtotal resection ofdisease is performed, the volume to receive IORT is determined by thesurgeon and radiation oncologist and marked with small hemoclips. Anintraoperative treatment applicator of appropriate size is chosen andthe energy selected. The uninvolved structures which cannot bephysically excluded from the treatment field are shielded secondarilywith malleable sterilized lead sheet cut-outs of a thickness appropriateto reduce the dose by 90%.

The anesthesia and surgery departments follow their standardtransportation policies for transporting the anesthetized patient, ifnecessary.

Major dose-limiting organs and tissues for IORT include small bowel,large bowel, stomach, spinal cord and nerve. Small bowel and stomach arealways be excluded or bypassed and large bowel whenever feasible.Tolerance of large bowel, nerve, vessels, bladder, bone and muscle isevaluated. Potential technical problems include difficulty in obtainingcomplete tumor bed coverage due to location on a pelvic sidewall,adherence to more than one pelvic location (i.e. sacrum plus lateralpelvic sidewall), narrow pelvis, and inferior location of a lesionmaking an abdominal approach difficult. The danger of beam obliquity andchoice of inadequate electron beam energy is inherent in all of thepreceding situations.

The major operative solutions include displacement of all or part of thedose-limiting organs and use of generous incisions to allow maximumflexibility in cone placement both via the abdominal and perinealapproaches. Small bowel or suture lines are be included in any IORTboosts but should be totally mobilized. A build up of fluid can occur inthe dependent pelvis so constant suction is maintained alongside thecone during treatment.

No anastomosis or diverting procedures are done prior to the IORT ifthis will bring the suture line into the radiation field. Spillage ofurine or bowel contents during the operative procedure is not acontraindication to IORT as these patients all have mechanical bowelprep preoperatively and perioperative antibiotic coverage.

Patients who have received preoperative EBRT of 45-50.40 Gy in 25-28fractions over 5-5½ weeks are scheduled for re-exploration and resection3-4 weeks from the date of completion of radiation therapy. The maximumallowable interval is 8 weeks. If the tumor or tumor bed can be includedwithin an IORT field the patient is randomized to receive an IORT boosteither with or without etanidazole. If IORT is not feasible, furtherexternal irradiation is given, if possible.

For patients with previous EBRT, resection is performed and theintraoperative portion of the irradiation is delivered at that time asabove, if possible. External beam irradiation, preferably 20-30 Gy plusPVI 5-FU, precedes surgery/IORT. Post operative irradiation begins 2-4weeks after surgery when indicated.

In situations where it is inappropriate to operate immediately, e.g.,initial re-resection outside parent institution, etc., re-operation forpurpose of delivering the intraoperative portion of irradiation can bedelayed up to 6 weeks from time of initial resection. If possible, lowdose external beam radiation plus CI 5-FU should precede the IORT boost.

INCORPORATION BY REFERENCE

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thecompounds and methods of use thereof described herein. Such equivalentsare considered to be within the scope of this invention and are coveredby the following claims. Those skilled in the art will also recognizethat all combinations of embodiments described herein are within thescope of the invention.

We claim:
 1. A method for the treatment of cancer in a patient,comprising: (a) administering to a patient with a locally advanced orrecurrent tumor a pharmaceutically acceptable composition comprising atherapeutically effective amount of a hypoxic cell radiation sensitizer;(b) after the administering of the hypoxic cell sensitizer to thepatient, performing resection of said tumor in an operating room,thereby leaving a body cavity; and (c) subjecting the body cavity to atherapeutically effective amount of intraoperative particle beamradiation therapy from a source, wherein said source of saidintraoperative particle beam radiation therapy is located in the sameoperating room and said intraoperative particle beam radiation therapyoccurs within one hour of administering said hypoxic cell radiationsensitizer, wherein said source is a mobile electron beam therapysystem.
 2. The method of claim 1, wherein the cancer is colon cancer,rectal cancer, stomach cancer, cancer of the small intestine, lungcancer, cervical cancer, brain cancer, pancreatic cancer, cancer of thehead or neck, esophageal cancer, breast cancer or cancer of the oralcavity.
 3. The method of claim 2, wherein the cancer is of the head orneck.
 4. The method of claim 2, wherein the cancer is rectal cancer. 5.The method of claim 2, wherein the cancer is lung cancer.
 6. The methodof claim 1, wherein the radiation sensitizer is etanidazole.
 7. Themethod of claim 1, wherein the radiation sensitizer is doranidazole. 8.The method of claim 1, wherein the patient is administered saidradiation sensitizer and subjected to said intraoperative particle beamradiation therapy within 40 minutes of each other.
 9. The method ofclaim 1, wherein said intraoperative particle beam radiation therapy isionizing.
 10. The method of claim 1, wherein said hypoxic cell radiationsensitizer is associated with a targeting moiety.
 11. The method ofclaim 10, wherein the targeting moiety is covalently associated with theradiation sensitizer.
 12. The method of claim 10, wherein the targetingmoiety is non-covalently associated with the radiation sensitizer. 13.The method of claim 10, wherein the targeting moiety and the radiationsensitizer are associated in a liposome.
 14. The method of claim 1,wherein said intraoperative particle beam radiation therapy isadministered in a single dosage.
 15. The method of claim 14, whereinsaid single dosage is selected from 5 to 20 Gy.
 16. The method of claim15, wherein said single dose is selected from 10 to 15 Gy.
 17. Themethod of claim 1, wherein said intraoperative particle beam therapy isadministered in two, three, four or five doses.
 18. The method of claim17, wherein said intraoperative particle beam therapy is administered intwo or three doses.
 19. The method of claim 1, wherein the patient isfurther administered a second form of radiation therapy selected frominternal radiation, external radiation and systemic radiation therapies.20. The method of claim 19, wherein said second form of radiationtherapy is selected from stereotactic body radiotherapy, stereotacticradiosurgery, and intensity-modulated radiation therapy.
 21. The methodof claim 19, wherein said second form of radiation therapy isadministered hours after said resection.
 22. The method of claim 19,wherein said second form of radiation therapy is administered one ormore days after said resection.
 23. The method of claim 19, wherein saidsecond form of radiation therapy is administered one or more hoursbefore said resection.
 24. The method of claim 19, wherein said secondform of radiation therapy is administered one or more days before saidsurgical resection.
 25. The method of claim 1, wherein said performingresection of said tumor comprises incising tissue to reveal said tumorand removing said tumor.
 26. The method of claim 25, wherein the methodfurther comprises step (d) wherein said tissue is sutured followingadministration of said intraoperative particle beam therapy.